Fungicidal compound and process of making same



G. HAGEMANN' ET AL 3,052,605

FUNGICIDAL COMPOUND AND PROCESS OF MAKING SAME Filed July 25. 1960 Sept. 4, 1962 A YTIFOYG/h'' (/4194 VIOLET pscnpun W WW w; in (H) ll vm'ra 's 0) f ase'nnlwv 6: 4. 0 Non/ms Luc/s Pamsse y Jean TEILLON Mmma Ase/Yr United States Patent 3,052,605 FUNGICIDAL COMPOUND AND PROCESS OF MAKING SAME Guy Hagemann, Vincennes, Seine, Gerard Nomin, Noisy-le-Sec, Seine, and Lucien Penasse and Jean Teillon, Paris, France, assignors, by mesne assignments, to Roussel-UCLAF, S.A., Paris, France, a corporation of France Filed July 25, 1960, Ser. No. 47,342 Claims priority, application France June 22, 1955 11 Claims. (Cl. 167-65) The present invention relates to a new fungicidal agent and, more particularly, to the fungicidal compound designated as Antifongine, and to a process of preparing the same by means of a biological process.

The present application is a continuation in part of copending applications Serial No. 746,560, filed July 3, 1958, and entitled Fungicidal Compound and a Process of Preparing Same, now abandoned, and Serial No. 592,258, filed June 19, 1956, and entitled New Antibiotic and Method of Preparing Same, now abandoned.

It is one object of the present invention to provide the highly effective fungicidal compound designated as Antifongine which is useful not only in the industrial field but also, more particularly, in the agricultural field where an effective protection against fungi and molds is desired.

Another object of the present invention is to provide a simple and efiective process of producing said fungicidal compound.

A further object of the present invention is to provide a new microorganism producing, on cultivating on a suitable nutrient medium, said new fungicidal compound.

Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.

The fungicidal compound Antifongine obtained according to the present invention is an organic compound which contains carbon, hydrogen, nitrogen, and oxygen. Under a polarizing microscope, the characteristic extinction pattern indicates that the new compound is crystalline. Anti- :fongine represents a yellow powder which, on heating, starts to decompose at 100 C.

Said Antifongine is very soluble in certain solvent mixtures but is of extremely low solubility in water, hydrocarbons, ketones, and chlorinated solvents. It is slightly soluble in lower alcohols and carbon disulfide, and is soluble in propylene glycol, isoquinoline, pyridine, triethylamine, and dimethyl formamide.

On microanalysis, the following values were found in a compound with an ash content of 1.3%: 63.6% C; 7.8% H; 2.8% N.

The results obtained on subjecting the Antifongine to various chemical reactions for its characterization are given in Table A.

TABLE A Reaction: Result Ninhydrin Negative. Benzidine Do. Tollens Do. 2,4-dinitro phenyl hydrazine. Do. Ferric chloride Do. Picric acid Do. Flavianic acid Do. Reinecke salt Do. Ceric nitrate Weak reaction. Hydroxylamine Amorphous precipitate.

The fungicidal compound Antifongine according to the present invention dissolves in the following concentrated acids: Sulfuric acid, phosphoric acid, hydrochloric acid, perchloric acid, and formic acid, thereby yielding an intense blue-green color which turns yellow on dilution ICC TABLE B (In angstroms) E} "13,, 2910 140 3450 340 3600 540 3800 764 4050 636 These maxima seem to indicate the presence of a polyene structure and most probably the structure of a conjugated heptaene.

FIG. 2 shows the infrared absorption spectrum of Antifongine which has transmission maximums at 5.85;, 6.12 14, 9.63 and 1316 absorption maximums at 6.27,u, 8.5 9.35 10.0/L, 11.28 and 11.82;; and shoulders at 6.40;, 8.85 9.10 11. and 1030a.

The fungicidal compound Antifongine has a high activity against various yeasts and molds. The said fungicidal compound shows also an interesting activity against phy-topathogenic fungi which cause infections on vegetables and fruit trees. It is highly active against the following microorganisms:

The following Table C gives the minimum inhibitory concentration (threshold value) in 'y/cc. determined on an agar containing culture medium:

TABLE C Microorganism: Threshold value in 'y/ cc. Saocharomyces cerevisiae L.41 0.01 to 0.25 Torula ufiliv 50 to Candida albicans 0.1 to 1 Botrytis allii 50 to 100 Botrytis roussillon 5 to 20 3 Calonectrz'a decora 1.0 Clitocybe tabescens 1.0 Colletotricham lindemuthianum 1.0 Cryptococcds neoformans" 1.0 Didymella lycopersici 1 to Uslilago avenae 1 Mucor microsporus 350 Choanephora cucurbit'arum 4.5 Cephalotecium roseum 35 Alternaria solani 1.0 Penicillium italicam 100 Fusariam caeruleam 100 Ustilago zeae 5O Monilia fructigena Therefore, it represents a valuable agent for the treatment of infections caused by such microorganisms.

A ntifongine diifers from trichomycin and candidin by its insolubility in water and slightly alkaline solutions wherein said trichomycin and candidin are soluble.

Antifongine diifers from nystatin, fungicidin, amphotericin A, and fradicin by the position of the maxima in its ultraviolet spectrum.

Furthermore, it differs from trichomycin, ascosin, and candicidin by its eifectiveness on Candida albicans. It prevents proliferation of said microorganism in a concentration of only 0.1 'y/cc. to 1.0 'y/cc.

The toxicity of crude Antifongine (E}Z" ,23O at 3800 angstroms) determined on mice, is about 3.5 mg./kg. when applied intravenously, 25 mg./kg. when applied subcutaneously, and at least 2000 mg./kg. when applied orally.

The chronic toxicity was determined on 4 groups of 10 male rats each, said rats weighing, on an average, 90 g. One group of rats served as control group; they received by means of a stomach sound an aqueous solution of the dispersing agent, such as a dispersing agent of the type sold under the trademark Tween. The other three groups received the same solutions having dispersed therein an amount corresponding to a dose of 10 mg./kg., 20 mg./kg., or, respectively, 50 mg./kg. of Antifongine also by means of a stomach sound. Said amounts of Antifongine were administered daily, except on Sundays,

for one month. The mortality of the animals was:

TABLE D Dose of fungicidal compound: Mortality Controls 5 of 10 animals. 10 mg./kg 2 of 10 animals. 20 mg./ kg Do. 50 mg./kg 3 of 10 animals.

In all cases the animals died due to endothoracic infections (pneumonia, pleurisy, pericarditis purulentis). Thus their death cannot be attributed to the Antifongine as is clearly shown from the larger number of deaths in the control group. No diiferences in the growth rate were observed betweencontrol animals and treated animals. Hematological'tests carried out before and after administration of Antifongine did not show any abnormality. Autopsy of the animals at the end of the treatment failed to show macroscopically any changes. Liver and kidney were investigated histologically without any abnormal finding. Thus the chronic toxicity of Antifongine is also quite'low.

Topical application of Antifongine in the form of a 5% ointment to the depilated skin of a guinea pig daily for 4 days did not show any irritation. Daily application for 6 daysto the mucous vulva of a female dog was well tolerated. Daily application for 5 days to the conjunctiva of a rabbit .causedmaximum irritation about 2 hours which almost completely disappeared after 24 hours. However, application ofthe ointment excipient alone produced the same irritating effects indicating that irritation is due to the excipient and not to Antifongine.

The particularly low toxity of Antifongine permits its utilization in veterinary medicine as agent against mycosis and the like diseases. For instance, when repeatedly applying Antifongine orally to mice infected with Candida albicans, the disease is attenuated and the life of the treated animals is considered prolonged.

When determining the in vitro effectiveness of Antifongine on the germination of conidia of Venturia inaequalis or apple scab, the crude Antifongine proves to be fungist-atic in a concentration of 250 'y/cc.

Furthermore, studies on the usefulness and eifectiveness of Antifongine on other phytopathogenic fungi shows the following results:

(a) It is highly effective against the spores of Alternaria oleracea and Glomerella cingalata; when testing according to the method of MacCallan, total inhibition of germination is found at a concentration of cc.

(1)) An interesting activity is found against Batrytis cinerea (gray mold rot of grapes), i.e. beginning inhibition of germination in a concentration of 100 'y/CC. and high effectiveness in a concentration of 500 'y/ cc.

(0) Very marked activity is observed against Dothichiza populea (causing a particular disease of the poplar tree) in a concentration of 500 7/ cc. and against Colletotrichum gramincaram (causing a particular fungus disease of corn) in a concentration of 100 'y/cc. to 500 'y/cc.

(d) Good protection against infection of vine leaves Iby lncinala necator (O'idium species) is afiorded by Antifongine on spraying vine leaves separated from the plant and kept alive with an aqueous suspension containing 1% of said compound.

When using in the experiments leaves removed from plants, a very remarkable activity on spot diseases in fruit trees such as caused by Ventaria inaeqaalis is observed. The infection is completely inhibited by the application of an aqueous suspension of the crude Antifongine in a concentration of 250 'y/Cc., even two weeks after the treatment.

No apparent signs of a phytotoxicity were observed in the various experiments when applying an agricultural preparation of the crude Antifongine to various plants, for instance, grapevine, fruit trees, potatoes, and tomatoes.

The agricultural preparation of Antifongine mentioned hereinabove consists of a powder of dried mycelium which contains about 1.5% of the crystalline Antifongine.

The crude Antifongine is an amorphous extraction product which contains about 30% of the crystalline Antifongine.

The antifungal activity of the Antifongine has been studied more particularly on Saccharomyces cerevisiae and on various clinical strains of Candida and has been compared with the activity of Nystatin as will be shown in the following tables.

(A) Saccharomyces cerevisiae.-The pH of the culture medium was 7.0.

(a) Saccharomyces ATCC 9763:

Under these conditions, Antifongine was five to six times more efiective on Saccharomyces than Nystatin. Apparently it is also more stable in the culture medium at 37 C. than Nystatin.

(b) Saccharomyces ATCC 9761:

was not too well tolerated when administered subcutaneously to mice in the form of an aqueous suspension of 2 mg./ cc. or 10 mg./cc., all further studies of the in vivo activity were made by oral administration. Antifongine was well tolerated even in amounts of 10 mg. per mouse.

(A) Infections with Candida albic-ans.--(a) Tests with mice: The mice were infected by intraperitoneal injections of a culture of Candida albicans (1 mg. of cells in 1 cc. of mucin). Antifongine was administered in the form of an aqueous suspension of a powder thereof, by means of the stomach sound once daily for 10 days. Parallel series of mice were treated in the same manner with Nystatin (purified powder Squibb with 3360 u./mg.). It may be mentioned that commercial Nystatin type Sifa has ordi- (B) Candida.-The pH of the culture medium was 7.0. narily an activity of only about 2000 u./mg. Trichomy- TABLE G Nystatin after- Antiiongine after- Type Strain Origin 18hrs. 36hrs. 48hrs. 96hrs. lShrs. 36hrs. 48hrS. 96hrs.

Candida albicans NRLL 7477 10 0.15

Candida albicans 4797 Candida albicans- Candida albicans C did nThir-mn Q Candida albicans Candida albicans- Candida albicans Candid nihirnn s Candida albicans Candida pseudotropicalis Candida pseudotropicalis Candida tropicalis Candida Masai-.. Toralopsis (Cryptococcas) neolCharge N0. 2116-144-3.

Charge No. 2313-191-I.

Charge N0. 2313-191-1.

formans. Torulopsis (Cryptococcas) neoformans.

}Charge No. 2313-65-V.

These tests show that the fungistatic activity of Antifongine against Candida is, on the average, six times greater than that of Nystatin after 18 hours. This difference remains the same after 36 hours and after 48 hours. After 96 hours the activity of Antifongine is, in general, somewhat inferior to that of Nystatin. This is apparently a real difierence between the two compounds and is due to their fungicidal activity as well as to their stability at 37 C. on prolonged contact for more than 48 hours with the culture medium. For practical purposes this difference is of no significant importance because an antifungal agent will ordinarily be completely eliminated after 48 hours.

The following tests prove that the fungistatic activity of Antifongine was not reduced in the presence of agents which are eflfective against bacteria. .These tests were carried out in vitro with several antibiotics. The results are given in the following Table H:

TABLE H [Minimum inhibitory concentration in 'y/cc. Antifongine sample 2116-144-3, culture medium pH: 7.0. Candida albicans NRRL-Y-477. Determination after 48 hours at 37 (3.]

Added Framy- Neomy- Tetra- Chlor- Terra- Streptoantibioties cetin cin cyclin amphenimycin mycin in ylce. base base col base base The results clearly demonstrate that Antifongine can be used with antibacterial antibiotics without afiecting its fungistatic activity.

Since preliminary tests had shown that Antifongine cin, the activity of which had been verified in vitro, did not shown any effectiveness in vivo.

The results of the various tests are compiled in the following Table I:

TABLE 1.

Comparative Activity of Antifongine and of Nystatin Against Candida albicans as Determined With Mice [This table shows the activity expressed in percent determined by mortality of the animals and extent of lesions] Daily dose, Test No. Antiiongine, Nystatin,

mg. percent percent 1 Charge No. 2116-131 (crude product). Charge No. 266940 (purified product). 3 Charge No. 2313-191-1 (purified product).

Although there are some variations due to diiferences in the seriousness of the experimental infection, it is evident that the activity in vivo of Antifongine is superior to that of Nystatin, when administered in equal doses; this superiority is very apparent when given in small doses as follows from Table I.

(b) Tests with rabbits: A method of investigating rabbits by exploring laparotomy was established which allows to determine the degree of infection by means of the number of visible micro-abscesses at the perirenal aponeurosis. It was found that Antifongine (Charge No. 2313-69411), purified E=795) when orally administered in doses of 20 mg. per kg. exhibits satisfactory activity against Candida albicans infections. Nystatin, when tested under the same conditions, has no noticeable effect and Trichomycin is too toxic even in doses of only 1 mg. per kg.

(B) Infections with Cryptococcus neoformans.-The infections were established by intraperitoneal injections of cultures of a pathogenic strain of Cryptococcus neoformans of the Pasteur Institute. This microorganism produces in mice septicemia and renal abscesses. With respect to medium serious or serious infections, the activity of Antifongine (Charge No. 2313-l91-I), when administered orally in daily doses of 2 mg. per mouse, Was 24% and 18%, respectively. Nystatin, under the same conditions did not exhibit any appreciable activity and respectively) in the same doses. In more moderate infections which cause, however, death of 50% of the test animals on the seventh day, the results were as given in the following Table K.

TABLE K Daily oral Antifongine, N ystatin, dose, mg. percent percent mortality mortality Thus noticeable activity of Antifongine is observed although a dose of 2 mg. is insufficient, while Nystatin is ineflfective.

(C) Various infections-While the above given tests were carried out in order to determine the in vivo activity of the Antifongine against various experimental mycoses, said compound was also tested for its activity against other pathogenic agents. However, no significant activity was observed.

(a) Activity against protozoa: Entamoeba histolytica: In vivo activity: 107 to 50 (less effective than Paromomyoin: 5 to 107). Trichomonas: No activity in an oral dose of 5 mg. per mouse.

(b) Influenza virus: No activity in an oral does of 5 mg. per mouse.

All these tests clearly prove that the new Antifongine is in vivo highly effective against various fungi. Its activity against Candida albicans is far superior to that of Nystatin, even after remaining in the culture medium for 48 hours, and against Cryptococcas neoformans. Its activity is not affected by the presence of antibacterial antibiotics.

In vivo Antifongine. has shown to be very effective on oral administration against systemic infections of mice by- Candida albicans. It exhibits also a very definite activity against infections by Cryptococcus neoformans. In these instances its effectiveness is superior to that of Nystatin.

In principle, the process of preparing Antifongine consists in growing a culture of Streptomyces paucisporogenes deposited with the American Type Culture Collection under N0. ATCC 12596 by aerobic immersed cultivation, for instance, according to the principle of the so-called multi-stage cultivation process, at temperatures preferably close to 30 C. The fermentation time varies between 48 hours and hours and is preferably about 70 hours. Aeration of the fermentors is preferably regulated in such a way that 0.5 volume to 2 volumes of air per volume of culture medium pass through said medium per minute.

The nutritive medium comprises nitrogen sources, such as corn steeping liquor concentrates, flours, distillery residues, yeasts; carbon sources, such as sugar, dextrines, starches; and different mineral salts which, serving as buffer agents, are indispensable for the growth of the cells. A culture medium which is particularly suitable for the production of Antifongine may, for instance, consist of the following ingredients:

Percent Dry corn steeping liquor 0.8 Soybean meal 2.5 Dextrin 1.3 Glucose 1.0 Calcium carbonate 0.2 Water 94.2

After fermentation is completed, the mycelium is filtered off. The filtrate is adjusted to a pH-value which is preferably close to 6.0, by the addition of acid and brought into contact with a cation exchange resin, such as, for instance, the cation exchange resin of the carboxylic acid-type synthetic resin sold under the trademark Amberlite IRC 50 (described in U.S.P. 2,340,111) in its sodium salt form in order to separate the antibiotic compound formed by the fermentation process.

The dried mycelium, obtained by filtration of the culture medium, is extracted by means of methanol containing calcium chloride, adding water to the extracts, evaporating the methanol, filtering off the precipitate, purifying it by chromatographic absorption, and recrystallizing Antifongine in a suitable solvent.

The filtered liquid culture medium which has been treated with the above mentioned cation exchange resin in order to extract the antibotic compound is evaporated to dryness and taken up in methanol. The solution is concentrated and extracted by means of butanol in the presence of water. The resulting butanol solution is concentrated, acetone and petroleum ether are added whereby a precipitate is obtained, a methanol solution of which is treated with a suitable ion exchange resin. The thus purified solution is concentrated and extracted by means of tertiary butanol. The crude Antifongine is obtained thereby. It may be recrystallized as described above.

According to a preferred embodiment of the present invention, Antifongine purified by chromatographic absorption on activated aluminum oxide which is eluted by a mixture of dioxane, water, and butyl acetate (40:52:18) to which 5% of acetic acid has been added. Antifongine is preferably recrystallized from a mixture of pyridine, dioxane, water, and butyl acetate The microorganism Streptomyces paucisoporogenes is a new species which differs, for instance, from Streptomyces rimosus in several respects and especially by its ability of producing not only an antibiotic but also the new fungicidal agent according to the present invention.

Streptomcyes paucisporogenes which produces these two active agents is a new species of the genus Streptomyces of the subdivision .Actinomycetes. It is found primarily in soil. It is isolated in form of pure cultures in accordance with conventional processes. Streptomyces paacz'sporogenes grows only poorly on solid culture media generally used for organisms of this type and results in colonies composed of filaments whose more or less branched end portion shows only exceptionally any growths of conidium chains.

It grows on complex orgamc agar media. On these media Streptomyces paucisporogenes is microscopically a small aerial mycelium formed of squat hyphae which carry spores only rarely. Most of the nitrogen sources, whether of ammoniacal, amine, or nitrate nature, favor development of an aerial mycelium. Development of an characterized by a yellowish-white aerial mycelium; the aerial mycelium is also improved by the addition to the felting of the vegetative mycelium is thick, sometimes agar or liquid culture medium of small amounts of mineral crazed. Occasionally, the organism secretes a small salts such as sodium chloride, magnesium sulfate, potasquantity of a soluble, yellow-brown pigment. The slum phosphate, as well as trace elements usually supplied mycelium develops particularly Well on a medium adby complex metabolites, such as soybean flour, liquors objusted to pH 7.5 and comprising a decoction, in one liter tained on steeping cereal grains, distillery by-products. of distilled water, of 200 g. of soy meal, g. of glucose, While the surface culture of Streptomyces paucisporo- 10 g. potato starch, and 20 g. of agar. genes is difiicult, submerged culture is easy, especially in Growth on chemically defined media (Czapeck, Conn, media containing a peptone, and is accompanied by a more etc.) is djlficult; there is, however, solubilization of calpronounced secretion of the soluble pigment. Milk is cium malate. 15 quickly coagulated and peptonized; on the other hand, the

On a natural substratum, such as potato slices bufsecretion of gelatinolytic diastases is very weak. fered to a pH of 7, Streptomyces paucisporogenes de- The following Table L shows the characteristic dilfervelops rapidly in the form of a crazed, cerebriiorm, ences between this new actinomycetes and other actinothick coating. However, mycelium growth is rare and mycetes which produce bas1c antibiotics.

TABLE L Aerial mycelium Antibiotic Nutritive Czapeck Potato Starch Aerial R d Actinomyces produced agar-medium agar-medium substrate agar-medium hyphae Milk tigrnfi 111 8. 8S

1 Albogriseolus Complex White becom- White to ash- Greyis h white Hydrolysis.-- Spirals neomycin. ing ashgrey. to pink.

grey. 2 Fradiae Neomycin Pink Sea-shellpink. Insmall do No spirals.

quantity. 3 Roseoflavus do White to White to None Grows abun- Spira1s. Coagulation pink. pinkish dantly. peptonizayellow. tion. 4 2103 Framycetin--- vio l a eous Roseatewhite- Hydro1ysis.-- do No coagulation p 5 Griseus Streptomycim Grey-green Grey-green White Stronghydrol- No spirals Coagulation ysis. pieptoniza- OH. 6 Bikiniensz'sd0 White Pale-grey Ochrous beige- Slight hydroldo Gradualhydrol- YSlS. YSIS. 7..-. Lavendulae Complex Lavender Wine-colored Black Hydrolysis Spirals"--- No coagulation streptopink. pink. mYClll. 8...- Vinaceus Viomycin.- Grey to grey- Moderate No spirals.

blue. hydrolysis. 9 Paucisporogenes Paromomy- Yellowish None-does Yellowgrey Nonedoes do Coagulation cin and white. not grow. not grow. peptonizaantifongine. tion.

of greyish-white color with yellow reflections, if present.

On a synthetic agar culture medium Sterptomyces paucisporogenes produces a rnycelium of lemon yellowish or clear beige color. The aerial mycelium is white or slightly pinkish and forms straight hyphae of a diameter Thus, the new microorganism Streptomyces paucisporw genes differs from known Streptomyces species as they are described, for instance, by Waksman et al., The Actinomycetes and Their Antibiotics, published, 1953, by Williams and Wilkins, on pages 11, 55, and 65, by the charof 1.5 to 2.011.. The fascicular branched portions are acteristic properties of the aerial mycelium shown in the very fine and have a diameter of 1.0;]. to 1.5;. The following Table M.

TABLE M Aerial mycelium Streptomyces Antibiotic Nutritive Ozapeck Potato Starch Aerial Reducspecies produced agar medium agar medium substrate agar medium hyphae Milk 1fiig n of a es 1 Streptomyces Erythro- Cream-- White Cream- Hydrolysis Spirals"--. Coagulationand erythreus. mycln. colored. colored peptonizabecoming tion. yellowish. 2 Streptomyces Not indicated. do Grey A few curl- I l flavogrixeus. mg tips. 3 Streptomyces Actinomycim- Yellowlsh to Grey No aerial No tips No coagulation '2 amibi ti s, yellowish mycehum. spirals. nor peptonlgreen. zation. 4..-- Streptomyces Paromomycin Yellowish- No aerial Yellowish N0 growth..- No spirals Coagulation paucisporoand antiwhite. mycellum; grey. and peptomgenes. fongine. does not zation.

grow.

end parts of said filaments often terminate in chains of 5 to 20 spherical or oval spores the diameter of which is the same as that of the supporting filaments. The dimensions of said conidia are about 1.0 to 1.5; X 1.011. 110 2.01.0.

On a complex organic agar medium containing yeast autolysate, Streptomyces paucisporogenes there develops It is clearly evident that the Streptomyces paucisporagenes species according to the present invention is a species carried out.

different from the known species.

The fluid culture media of Streptomyces paucisporogenes exhibit also antibiotic activity which increases with the pH of the agar on which the potency control test is This shows that the new antibiotic belongs 1 1 to the group of basic antibiotics: streptomycin, streptothricin, neomycin, framycetin, but the indications supplied by a so called crossed antibiosis test point to essential differences which exist between Streptomyces paucisorogenes 12 Benedict for classifying Streptomycetes according to selected groups (see Applied Microbiology, January 1958, vol. 6, pages 52 to 79'). The criterion used by Pridham et al. for diiferentiation of Streptomyces species in the and those microorganisms which produce the above-named morphology of the sporophores of mature cultures. antlblotlcs (Table S. A. Waksman uses a different method of differentiation in his book The Actinomycetes and Their Anti- TABLE N biotics, published by Williams and Wilkins, Baltimore, Sensitivity of Difierent Streptomyces to the Principal Md, 195 3, namely the microscopical and macroscopicai Antibiotics comparison of the fungi, cultivated at the same time and 0:11 seqsmve +=Fair1y Sen'sitive under the same conditions on a series of agar-containing =l==Ve Yht e se t sensltlve nutrient media as they are conventionally used in rmcrobiology. Pauci- Lavm- Antibiotics sporo- Griseus time Fradiae 2103 h followmg 3? 0 comparauve results 937133 cultivation of the original strain of Streptomyces paucisporogenes and of a sporulated variant in comparison Streptomycin 0 with Streptomyces rimosus forma paromomycinus P 57 Neomyein-- i a: 0 0 giomyciltku i I if with respect to the following criteria.

ramyce 111.... :l: :l: Erythromycin 0 (1) Growth. %ure;myci1li1. i i (2) Vegetative mycelrum 61'! mYG Chloramphenicoh i Aerlal mycehum Paromomyein 0 =1: :l: :I: (4) Spores, and

(5) Pigrnentation of the medium TABLE 0 Criteria S. paucisporogeites S. paucisporogems S. rimosus forma paramoorigin sporulated form mycmus Non-specific culture medium:

zape 1 No growth Slight+ :l:.

2 Colorless. 3 0. 4 0. 5 0. Potato 1 2 Light yellow Light yellow... Translucent. 2 f\lVhite, very scanty... White, scanty 0. 0. 5 0 0. Yeast extract 1 2 Light maroon Light yellow. 3 Light grey abundant" 0. 4 0 0. 5 0. Meat extract 1 2 Translucent. 3 0. 4 0. 5 N Z amine 1 2 Dark maroon- Pale yellow. 2 Grey beige scanty grackled, white, scanty.

0 5 Very intense maroon. 0. Specific culture media:

Dextrin-calcium No. 253 1 2 Light m'aroon-... Yellowish brown Light maroon. 3 Dark grey, scanty Abundant, pinkish beige Scanty, light grey. g. 0 i Malt calcium extract N o. 265... 1

2 Light beige. 3 White, traces. 4 0. 5 0. Com-steep lactose No. 275 B 1 2 Colorless. 3 Scanty, white. 4 0 0 0. 5 o n 0.

parative tests were carried out according to the method suggested by T. G. Pridham, C. W. Hesseltine, and R. G.

The first five culture media which are designated as non-specific media are those used by various authors for characterization and differentiation.

I. COMPARISON OF THE SPOROPHORES ON AN OPTIMUM CULTURE MEDIUM (a) The original strain of Streptomyces paucisporogenes does not sporu'late on any of the tested culture media. This negative chanacteristic distiguishes the strain clearly from.- all other species of Streptomyces described heretofore. (b) Its sporulating van'ant shows abundant sporulation on the calcium-malt extract medium No. 265.

13 14 (c) S. rimosus forma paromomycinus and S. paucisporo- Potatoes:

genes (sporulating variant) were compared on their Potato 200 optimum culture media. Glucose 20 The following Table P shows the results obtained Agar 2 thereby Meat extract:

TABLE P Glucose Heart extr 5 Hmomiorma Pammomwmw g gg ggwggfi Soybean peptone 5 10 Calcium carbonate 1 Dextrin-caleium medium (opti- Malt extract-calcium medium (op- Sodmm chlonde 5 mumculture medium) No. 253. timum culture medium) No. 265. Agar 20 Non-verticillated sporophores, ir- Verticillated sporophores generally Dextrin-calcium 253;

regular branching. 2 branches per verticil. D extri n I 10 Lateral branches coiled to spirals Lateral branches coiled to spirals C t li uor 1 or agglomerated. or agglomerated. S 0111 5 Long chains of spores Short chains of spores. A o i lf t 10 According to Pridham: Sodium nitrate 2 SectionSpira. Sectionmonoverticillus-spira." Zi lf ()3 Agar 20 Czapeck: Dipotassium phosphate 1 Saccharose 5 Calcium carbonate 10 Sodium nitrate 2 Sodium chloride 3 Dipotassium phosphate 1 Magnesium sulfate 0.02 Potassium chloride 0.5 Ferrous sulfate 0.03 Magnesium su1fate.H O 0.5 Manganous sulfate 0.007 2 13; sulfate 2 The following Table Q shows the difierences between the species Streptomyces paucisporogenes and Strepto- Yeast extract: myces rimosus forma paromomycinus NRRL 2455 on Glucose 5 other agar-containing culture media. In said table, A Yeast extract 10 indicates Streptomyces rimosus andB Streptomyces pauci- Dipotassium phosphate 0.25 sporogenes.

TABLE Q Vegetative mycelium Aerial mycelium Soluble pigment Agar containing culture medium A B A B A B Glycerol-asparagine Light yellow to Grey-15h White No mycelium None formed None formed.

light brown. formed. Syntheticstarch Light brown Feeble growth White, slight do "do Faintlymaroon formation. colore Calcium malate Lightbrownishyellow, do hite Greyish d0 Maroon. Agar nutrient medium Yellowish orange to YelloWish white Little or no Light maroon to do Do.

light brown. firsilum greyish. Glucose tryptone Light yellow to Greyish beige.-." White". Whitish beige Faint light brown Light maroon.

light brown. coloration.

Magnesium sulfate.H O 0.25 The cultures of the two species of Streptomyces exhibit Agar 25 the following characteristic features: N Z amine; A. Streptomyces rimosus forma paromomycinus NRRL Potato 100 2455.The surface colonies are raised, smooth, wrinkled N Z amine A 10 or plaited, and cracked at the regions of intense growth. Agar Often the agar itself is cracked. Under the microscope M alt ca1cium 26$ 5 the aerial hyphae are irregularly branched. The lateral branches are short and coiled. Numerous spirals are Malt F 30 present extending over the greater part of the medium and Amfmmulll sulfate 5 are frequently found in dense agglomeration. The ter- Sodmm. mtrate 1 minal parts of the aerial hyphae are subdivided in chains Magnesmm sulfate of spores. Litmus milk is ordinarily not peptonized. Ferrous Sulfate 0'015 B. Strepromyces paucisporogenes.The surface colot q Sulfate 0015 nies are covered with a beige, discrete, readily antolyzed Dlpotasslum Phosphate aerial mycelium. The mycelium is composed of long Calcium carbonate 5 entangled filaments, which can be parted with difiiculty P chlonde only. Under the microscope long, tin, only slightly Z ulfate 0.015 branched hyphae are observed which, even when young, Agar rapidly undergo lysis to a large extent. There are no Corn-steep lactose: spirals. Propagation of the mycelium is achieved by Lactose 5 budding of portions of the filaments which have not un- Corn-steep liquor 5 dergone lysis. Sporulation which is always very feeble, Ammonium sulfate 0.15 proceeds only under very favorable circumstances and Calcium carbonate 0.5 on especially adapted culture media. Agar 25 There are also diflerences in the utilization of carbo- Dipotassium phosphate 0.4 hydrates. Both microorganisms utilize the carbohydrates Magnesium sulfate 0.4 in a synthetic agar medium according to Pridham and TABLE R Streptomyces rimosus Streptomyces paucisporo- Carbohydrate forma paromomycinus genes Glucose Lactose Saceharose The following examples serve to illustrate the present invention without, however, limiting the same thereto. More particularly, the reaction time and temperature, the nature of the reagents and the solvents may be varied by those skilled in the art in accordance with the principles set forth herein and in the claims annexed thereto.

EXAMPLE 1 Shaker Fermentation The following materials are used to prepare an aqueous fermentation medium:

G. Glycerol 10 Bacteriological peptone 18 Corn steeping liquor 3 Sodium chloride 4 Calcium carbonate 1 With distilled water made up to 1000 cc.

200 cc. portions of the medium are filled into Erlenmeyer flasks which are then sterilized at 121 C. for thirty minutes. The flasks are cooled and inoculated with an aqueous suspension of an ATCC 12596 strain of Streptomyces paucisporogenes obtained as surface growth on a nutrient agar medium. The flasks are kept for 7 days at 30 C. on an oscillating shaker (80 oscillations per minute; extent of oscillation7 cm.).

EXAMPLE 2 Semi-Industrial Aerobic Fermentation The following materials are used to prepare an aqueous fermentation medium:

G. Peanut flour 26 Lumped sugar 13 Dextrin 10 Sodium chloride Calcium carbonate 2 With distilled water made up to 1000 cc.

previously sterilized:

Percent Soybean meal 2.5 Corn starch 1.3 Lumped glucose 1.0 Calcium carbonate 0.2 Tap water 95.0

The seeded wort is vigorously shaken and aerated by injecting sterile air at the rate of 350 liter per minute and is kept fermenting for 70 hours at 30 C.

15 EXAMPLE 3 Preparation of the Product Containing the Fungicidal Compound Antifongine for Use in Agriculture '100 liters of a culture obtained according to Examples 1 or 2 are stirred with 1 kg. of diatomaceous earth. The homogeneous mixture is filtered through a filter press 'by means of compressed air. The filter press cloth has previously been treated with an aqueous suspension of diatomaceous earth. 18 kg. of a moist filter cake are obtained. The filter cake is placed on and uniformly distributed over trays and is dried in a vacuum chamber at a vacuum of 25 mm./Hg by heating the trays to 45 C. or drying is efiected in a ventilated drying oven heated to 50 C. Drying is completed in about 15 hours. The dried product is pulverized in an impact pulverizer or hammer mill, for instance, of the Forplex type provided with classifier, and is then finely ground in a cylindrical homogenizer rnill until a very fine homogeneous powder of a grain size of less than 25,11. is obtained. The yield is 5 kg. of the agricultural product containing the fungicidal compound Antifongine.

EXAMPLE 4 Preparation of the Crude Fungicidal Compound Antifongine From the Mycelium of a Culture of Streptornycespaucisporogenes (AT CC 12596) Extract No. 1 0.96 Extract No. 2 2.04 Extract No. 3 0.99 Extract No. 4 1- 0.68 Extract No. 5 0.47 Total amount 5.14

The total amount of the resulting precipitate has a fungicidal activity against Saccharomyces cerevisiae in a dilution of 0.1 7/66. and against Candida albicans in a dilution of 0.1 'y/cc. to 1.0 'y/cc. The compound shows an UV. absorption intensity of E}?,, =230 at 3800 angstroms in a mixture of methanol, acetic acid, and water (722:1).

EXAMPLE 5 Purification of the Crude Fungicidal Compound Antifongine and Preparation of Amorphous Fungicidal Compound Antifongine 20 g. of crude fungicidal compound Antifongine obtained according to Example 4, are dissolved in a mixture of 120 cc. of dioxane, 90 cc. of pyridine, 54 cc. of butyl acetate and 156 cc. of water. Undissolved matter is filtered off. The solution is passed over a column of 600 g. of activated aluminum oxide which is then washed with 1045 cc. of the same solvent mixture until the first yellow colored inactive layer which moves rather rapidly has disappeared. The main fraction is eluted by means of 1060 cc. of a mixture of dioxane, water, and butyl acetate (40:52: 18) containing 5% of acetic acid. The resulting eluate is poured into a mixture of 10 liters of ether and 1 liter of methanol. The precipitated compound is filtered off. 6.5 g. of an amorphous yellow compound are obtained. The U.V. absorption intensity of said compound is lfin; 585 at 3800 angstroms EXAMPLE 6 Purification f the Amorphous F ungic'idal Compound Antifongine Ei? =736 at 3800 angstroms in aqueous methanol containing acetic acid. The compound is recrystallized twice from the same solvent mixture. 8.4 g. of the crystalline Antifongine are finally obtained having an U.V. absorption intensity of This new compound represents a yellow powder which shows the characteristic extinction pattern of a crystalline compound under a polarization microscope. The crystals have an extremely low solubility in water, hydrocarbons, ketones, and chlorinated solvents. They are slightly soluble in lower alcohols and in carbon disulfide and soluble in propylene glycol, isoquinoline, pyridine, triethylamine, and dimethylformamide.

Analysis.-63.6% C, 7.8% H, 2.8% N, 1.3% ash.

EXAMPLE 7 Preparation of Antifongine From the Liquid Filtrute of a Culture of Streptomyces paucisporogenes (ATCC 12596) The culture medium obtained according to Examples 1 or 2, is centrifuged and filtered. The pH-value of the filtrate is adjusted to a pH of 6.0 by the addition of dilute (1:1) sulfuric acid. The solution is treated with Arnberlite IRC 50" (U.S.P. 2,340,111) which previously had been treated with N sodium hydroxide solution and washed with distilled water until neutral.

130 liters of the resulting resin-treated solution are evaporated to dryness in a vacuum. The residue is extracted three times, each time with 10 liters of methanol. The methanolic solution is concentrated under reduced pressure to 500 cc. Two liters of water are added. The mixture is extracted 4 times, each time with 1 liter of ,butanol. The combined extracts are concentrated under reduced pressure to 300 cc. and are filtered. Ten times the volume of the concentrated extracts of a mixture of acetone and petroleum ether (boiling range: 3060 C.) in the'proportion of 1:2 is added. The precipitate is filtered off. 11.3 g. of crude Antifongine are obtained.

It shows a threshold value (minimum inhibitory effect of 0.1 'y/ cc. with respect to Saccharomyces cerevisiae and a threshold value of 0.1 'y/ cc. to 1.0 'y/cc. on Candida albican in an agar medium.

EXAMPLE 8 Purification of the Crude Antifongine g. of the crude Antifongine prepared according to the preceding Example 7, are dissolved in a mixture of 250 cc. of methanol and 125 cc. of water. The solution is passed over a column of 10 g. of the acidic form of a sulfonyl group-containing cation exchange resin known to the trade as Amberlite IR 120 (U.S.P. 2,340,111) and then over a column of 20 g. of the hydroxylated form of the anion exchange resin known to the trade as Amherlite 1R 4 B (U.S.P. 2,591,573). The purified solution is concentrated in a vacuum, taken up with100 cc. of tertiary butanol at 35 C. and evaporated to dryness by lyophilizing. 3.5 g. of purified Antifongine are obtained. Said product may further be purified by chromatographic 18 absorption and recrystallization as described in detail in Examples 5 and 6.

EXAMPLE 9 Preparation of an Emulsifiable Concentrate for Agricultural Purposes 1 g. of crude Antifongine,

10 g. of ethoxy ethanol,

500 mg. of calcium chloride,

10 mg. of 2,6-di-(tertiary butyl)-p-cresol, and

mg. of a polyoxy ethylene substituted alkyl phenol are intimately mixed with each other and the mixture is diluted with 5 l. to 10 l. of water. Thereby, "a stable suspension is obtained which is applied by spraying and which is effective against Venturia inaequalis, Botrytis, Oidium, and the like fungi.

EXAMPLE 10 Preparation of a Fungicidal Powder for Agricultural Use The crude Antifongine is dissolved in alcohol which may contain calcium chloride. An antioxidant of the tertiary butyl phenol type, for instance, 2,6-di-(tertiary butyl)-p-cresol, is added in an amount of 0.01% to 1.0%. The resulting solution is sprayed over an inert powder, such as calcium carbonate, talc, clay, or dried silica gel. The mixture is dried at a low temperature in a vacuum and the dried product is ground mechanically to yield a powder with a grain size of about 10 This powder is diluted by mixing it with a finely powdered inert carrier of about the same density in such a way that the resulting mixture contains about 20 g. of the crude Antifongine per kg. This powder is applied in amounts of about 25 kg. per hectare.

Of course, the crude Antifongine as well as the agricultural product obtained from the mycelium according to Example 3 may be converted not only into powders and emulsifiable preparations of Examples 9 and 10 but also into other preparations, such as solutions in propylene glycol or mixtures of solvents which may be applied by spraying to the plants to be treated. Ordinarily, it is not necessary to use the purified amorphous compound of Example 5 or the crystalline compound of Example 6. Such purified compounds, however, may be useful in animal therapy for the treatment of fungus infections.

Separation of Paromomycin from the culture filtrate is described more in detail in copending application Serial No. 592,258, filed June 19, 1956, and entitled New Antibiotic and Method of Preparing Same.

We claim:

1. The fungicidal compound Antifongine produced by cultivating Streptomyces paucisporogenes on an aqueous medium containing minerals and assimilable sources of carbon and nitrogen and recovering Antifongime halving the following characteristics:

(1) The compound being composed of carbon, hydro gen, nitrogen, and oxygen;

(2) analysis of said compound showing a product containing about 1.3% of ash;

about 63.6% C; about 7.8% H; and about 2.8% N;

(3) being effective against fungi and yeasts including phytopathogenic fungi;

(4) having a negative ninhydrin, benzidine, Tollens, 2,4-dinitro phenyl hydrazine ferric chloride, picric acid, and Reinecke salt tests and having a positive ceric nitrate test;

(5) giving with hydroxylamine an amorphous precipitate;

(6) being soluble in propylene glycol, isoquinoline,

pyridine, triethylamine, and dimethylformamide;

(7) being slightly soluble in lower alkanols and carbon disulfide;

(8) having an extremely low solubility in water, hydrocarbons, ketones, and chlorinated solvents;

(9) dissolving in concentrated sulfuric acid, phosphoric acid, hydrochloric acid, perchloric acid and formic acid, thereby yielding an intensely blue or green solution which, on dilution with water, changes its color to yellow;

(10) the ultraviolet spectrum of a solution in aqueous methanolic acetic acid (proportion: 7 parts by volume of methanol to 2 parts by volume of acetic acid to 1 part by volume of water) having the following maxima and extinction values:

Maxirna in angstroms:

(11) having a characteristic infrared spectrum having transmission maximums at 585 6.12,u, 9.63,u and 13.16 t; absorption maximums at 6.27,, 8.5 9.35,, 10.0w, 1128 and 11.82,:t; and shoulders at 6.40 8.8510, 9.10,u and 1030p;

(12) having substantially no phytotoxicity;

(13) said compound in substantially pure state being a yellow crystalline powder; and

(14) starting to decompose, on heating, at 100 C.

2. A composition for combatting phytopathogenic fungi comprising Antifongine of claim 1 and an inert carrier.

3. A composition for combating phytopathogenic fungi comprising, as active fungicidal agent, Antfongine according to claim 1, and an aqueous emulsion base, said fungicidal compound being emulsified in said emulsion base.

4. A composition for combating phytopathogenic fungi comprising, as active fungicidal agent, the finely pulverized Antifongine according to claim -1, and a finely pulverized inert carrier, said fungicidal compound being intimately mixed with and uniformly distributed throughout said inert carrier.

5. The process of combating phytopathogenic fungi, said process comprising applying to plants affected by such phytopathogenic fungi a composition containing Antifongine according to claim 1.

6. In a process of preparing Antifongine, the steps comprising extracting a mycelium obtained from a culture of Streptomyces paucisporogenes with methanol containing calcium chloride, adding water to the extracts,

20 distilling 05 methanol, filtering oil the precipitate, and purifying said precipitate by chromatographic absorption and recrystallization.

7. The process according to claim 6, wherein Antifongine is purified by chromatographic absorption on activated aluminum oxide and wherein a mixture of dioxane, water, and butyl acetate in the approximate proportion of 40:52:18, said mixture containing 5% of acetic acid is used as eluting solvent.

8. The process according to claim 7, wherein Antifongine is recrystallized from a mixture of pyridine, dioxane, water, and .butyl acetate in the approximate proportion of 30:40:52118.

9. In a process of preparing Antifongine, the steps comprising evaporating to dryness a filtrate of a culture of Streptomyces paucisporogenes substantially free of parornomycin formed during fermentation, taking up the residue with methanol, concentrating the solution, adding water to the solution, extracting the resulting mixture with butanol, concentrating by evaporation the :butanol solution, adding thereto a mixture of acetone and petroleum ether, filtering off the precipitate, treating the precipitate with ion exchange resins in aqueous methanol, concentrating the thus treated and purified solution, extracting the concentrate by means of tertiary butanol, and purifying the crude Antifongine isolated from the butanol solution by chromatographic absorption and recrystallizaion.

10. The process according to claim 9, wherein Antifongine is purified by chromatographic absorption on activated aluminum oxide and wherein a mixture of dioxane, water, and butyl acetate in the approximate proportion of 40:52:18, said mixture containing 5% of acetic acid is used as eluting solvent.

11. The process according to claim 10, wherein Antifongine is recrystallized from a mixture of pyridine, dioxane, Water, and butyl acetate in the approximate proportion of 30:40:52z18.

References Cited in the file of this patent UNITED STATES PATENTS Lindefelser et al Dec. 23, 1958 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No 3,052,605 September 4. 1962 Guy Hagemann et alo It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 6, line 40, for "shown" read show column 413, after "TABLE P" line 20, insert the following Composition of the culture media used in these tests The amounts given in g. are those per 1000 g of culture medium, The difference between the given amounts and 1000 g. is made up with water,

Signed and sealed this 16th day of April 1963.

(SEAL) Attest:

ERNEST W. SWIDER DAVID L. LADD Attesting Officer Commissioner of Patents 

1. THE FUNFICIDAL COMPOUND ANTIFONGINE PRODUCED BY CULTIVATING STREPTOMYES PAUCISKPOROGENES ON AN AQUEOUS MEDIUM CONTAINING MINERALS AND ASSIMILABLE SOURCES OF CARBON AND NITROGEN AND RECOVERING ANTIFONGINE HAVING THE FOLLOWING CHARACTERISTICS: (1) THE COMPOUND BEING COMPOSED OF CARBON, HYDROGEN, NITROGEN, AND OXYGEN; (2) ANALYSIS OF SAID COMPOUND SHOWING A PRODUCT CONTAINING ABOUT 1.3% OF ASH; ABOUT 63.6% C; ABOUT 7.8% H; AND ABOUT 2.8% N; (3) BEING EFFECTIVE AGAINST FUNGI AND YEASTS INCLUDING PHYTOPATHOGENIC FUNGI; (4) HAVING A NEGATIVE NINHYDRIN, BENZIDINE, TOLLENS, 2,4-DINITRO PHENYL HYDRAZINE FERRIC CHLORIDE, PICRIC ACID, AND REINECKE SALT TESTS AND HAVING A POSITIVE CERIC NITRATE TEST; (5) GIVING WITH HYDROXYLAMINE AN AMORPHOUS PRECIPITATE; (6) BEING SOLUBLE IN PROPYLENE GLYCOL, ISOQUINOLINE, PYRIDINE, TRIETHYLAMINE, AND DIMETHYLFORMAMIDE; (7) BEING SLIGHTLY SOLUBLE IN LOWER ALKANOLS AND CARBON DISULFIDE; (8) HAVING AN EXTREMELY LOW SOLUBILITY IN WATER, HYDROCARBONS, KETONES, AND CHLORINATED SOLVENTS; (9) DISSOLVING IN CONCENTRATED SULFURIC ACID, PHOSPHORIC ACID, HYDROCHLORIC ACID, PERCHLORIC ACID AND FORMIC ACID, THEREBY YIELDING AN INTENSELY BLUE OR GREEN SOLUTION WHICH, ON DILUTION WITH WATER, CHANGES IT COLOR TO YELLOW; (10) THE ULTRAVOILET SPECTRUM OF A SOLUTION IN AQUEOUS METHANOLIC ACETIC ACID (PROPORTION: 7 PARTS BY VOLUME OF METHANOL TO 2 PARTS BY VOLUME OF ACETIC ACID TO 1 PART BY VOLUME OF WATER) HAVING THE FOLLOWING MAXIMA AND EXTINCTION VALUES: MAXIMA IN ANGSTROMS: 